Non-splicing variants of gp350/220

ABSTRACT

Compositions comprising gp350 variant DNA and amino acid sequences are provided, as are vectors and host cells containing such sequences. Also provided is a process for producing homogeneous gp350 protein recombinantly and in the absence of production of gp220 protein, pharmaceutical compositions containing such protein and prophylactic treatments making use of such proteins.

TECHNICAL FIELD

This invention relates to methods for making and using and compositionscontaining Epstein Barr virus (EBV) gp350 DNA and protein sequences.

BACKGROUND

Epstein-Barr virus (EBV), a member of the herpesvirus group, causesinfectious mononucleosis in humans. The disease affects more than 90% ofthe population. Health analysts estimate the cost of the disease in theUnited States is 100 million dollars per year. The virus is spreadprimarily by exchange of saliva from individuals who shed the virus.Children infected with EBV are largely asymptomatic or have very mildsymptoms, while adolescents and adults who become infected developtypical infectious mononucleosis, characterized by fever, pharyngitis,and adenopathy. People who have been infected maintain anti-EBVantibodies for the remainder of their lives, and are thus immune tofurther infection. Currently there is no commercially available EBVvaccine.

In addition to its infectious qualities, EBV has been shown to transformlymphocytes into rapidly dividing cells and has therefore beenimplicated in several different lymphomas, including Burkitt's lymphomaand oral hairy leukoplakia. EBV has also been detected in tissue samplesfrom nasopharyngeal tumors. Worldwide it is estimated that 80,000 casesof nasopharyngeal cancer occur and it is more prevalent in ethnicChinese populations.

Development of a live, attenuated vaccine for EBV has been and still isproblematic. Because of the potential oncogenic nature associated withEBV, researchers have been reluctant to use a live vaccine approach.This invention overcomes the problems associated with live vaccinedevelopment by creating methods and compositions for a subunit vaccine,that does not require the use of a potentially oncogenic live virus. Asubunit vaccine uses one or more antigenic proteins from the virus thatwill elicit an immune response and confer immunity.

Two of the more important antigenic EBV proteins are glycoprotein(s)gp350/300 and gp220/200 that form part of the viral membrane envelopeand allow virus particles to bind to and enter human target cells byinteracting with the cellular membrane protein, CD21. See Nemerow, J.Virology 61:1416 (1987). They have long been singled out as subunitvaccine candidates but difficulties in obtaining antigenically activeprotein purified from native sources and low yields from recombinantlyproduced sources have hampered efforts of researcher and vaccinedevelopers. In the literature these proteins are referred to using avariety of molecular weight ranges (350 or 300 kilodaltons (kD) for oneof the proteins and 220 or 200 kDs for the other protein). The gp350 or300 protein is herein referred to as gp350 protein and the gp220 or 200protein is herein referred to as gp220 protein. Collectively, bothproteins are herein referred to as gp350/220 protein(s).

An alternatively spliced, single gene encodes the gp350/220 proteins andresults in the generation of gp350 and gp220 mRNA transcripts; nonaturally occurring variations in the gp350/220 gene splice sites areknown. The gene produces two expression products, the gp350 and gp220proteins. The open reading frame for the gp350/220 DNA sequence is 2721base pairs (bp). The entire reading frame encodes the 907 amino acids ofgp350. See U.S. Pat. No. 4,707,358 issued to Kieff (1987). The splicedversion of the reading frame covers 2130 bases and translates into gp220protein, a 710 amino acid sequence. The theoretical molecular weights ofgp350 protein and gp220 protein are 95 kD and 70 kD, respectively. Themeasured molecular weights of expressed gp350 protein and gp220 proteinvary but are approximately 350 kilodaltons and 220 kilodaltons (kD),respectively. The extensive glycosylation of the proteins accounts fordifference between the predicted and actual molecular weights. In anyone cell, both gp350 and gp220 proteins are produced at a molar ratioranging from about 6:1 to 1:1. For example, in B95-8 cells, which arepersistently infected with EBV, the ratio appears to vary but sometimesapproaches the 6:1 range. See, Miller, Proc. Natl. Acad. Sci. 69:383(1972).

Similarly, recombinant production of these glycoproteins has heretoforeusually resulted in a mixture of gp350 and gp220 protein being produced.Heretodate, the gp350/220 proteins have been expressed in rat pituitary,Chinese hamster ovary VERO (African green monkey kidney) cells, as wellas in yeast cells. See, Whang, J. Virol. 61:1796 (1982), Motz, Gene44:353 (1986) and Emini, Virology 166:387 (1988). A bovinepapillomavirus virus expression system has also been used to makegp350/220 proteins in mouse fibroblast cells. See, Madej, Vaccine 10:777(1992). Laboratory and vaccine strains of Vaccinia virus have also beenused to express gp 350/220 proteins. Modified recombinant versions ofthe EBV gp350/220 DNA and protein are known in the art. Specifically,recombinant truncated constructs of the gp350/220 gene lacking themembrane spanning sequence have been made. Such constructs still producea mixture of the two gp 350 and gp220, but deletion of the membranespanning region permits secretion of the proteins. See, Finerty, J. Gen.Virology 73:449 (1992) and Madej, Vaccine 10:777 (1992). Also, variousrecombinantly produced restriction fragments and fusion proteinscomprising various gp350/220 sequences have also been made and expressedin E. coli. See EP Patent Publication 0 173 254 published Jul. 24, 1991.

Accordingly, EBV research relating to gp350/220 heretodate has focusedeither on obtaining efficient expression of the native gp350/220sequence or on a modified sequence lacking the transmembrane domain,resulting in a mixture of the two alternate spliced versions of thenative or transmembrane lacking protein, or on production of epitopicfragment sequences in β-galactosidase fusion proteins.

Partially purified preparations of gp350/220 are known. See, Finerty, J.Gen. Virology 73:449 (1992) (recombinantly produced, partiallypurified). With respect to native gp350/220 protein, in most instances,the purification procedures resulted in inactivating the antigenicity ofthe protein, making it unacceptable for use in a subunit vaccine.However, highly purified preparations of antigenically active gp350protein from native (i.e., non-recombinant) sources have been reportedin the scientific literature. See, David, J. Immunol. Methods 108:231(1988). Additionally recombinant vaccine virus expressing gp350/220protein was used to vaccinate cottontop tamarins against EBV-inducedlymphoma. See, Morgan, J. Med. Virology 25:189 (1988), Mackett, EMBO J.4:3229 (1985) and Mackett, VACCINES '86, pp 293(Lerner R A, Chanock R M,Brown F Eds., 1986, Cold Spring Harbor Laboratory). However, the viralgp350/220 DNA sequence has not heretofore been engineered so as toenable expression solely of either one of the alternate spliced versionsof the gene, thereby enabling and ensuring the production of pure gp350or gp220 protein. Nor has a recombinant or mutant virus been made thatexpresses one or the other of the gp350 or gp220 proteins.

Generally, splice sites facilitate the processing of pre-mRNA moleculesinto mRNA. In polyoma virus, splice sites are required for the efficientaccumulation of late mRNA's. Alteration of the 3′ and 5′ splice sites inpolyoma virus transcripts decreased or completely blocked mRNAaccumulation. See, Treisman, Nature 292:595 (1981). In SV40 virus,excisable intervening sequences facilitate mRNA transport out of thenucleus and mRNA stabilization in the nucleus and because theseintron/exon junction sequences facilitate binding of small, nuclear, RNPparticles, it is thought that prespliced mRNA's might fail to associateproperly with processing pathways. It has been shown that pointmutations at exon/intron splice sites reduce exon/intron cleavage andcan disrupt pre-mRNA processing, nuclear transport and stability. See,Ryu, J. Virology 63:4386 (1989) and Gross, Nature 286:634 (1980).

Therefore, until the present invention, the effect of splice sitemodification on the functional expression and antigenic activity of theproteins encoded by the EBV gp350/220 sequence was at best unknown andunpredictable.

Additional background literature includes the following. EBV biology anddisease is generally reviewed in Straus, Annal of Int. Med. 118:45(1993). A description of the EBV BLLFI open reading frame is found inBaer, Nature 310:207 (1984). Descriptions of the Epstein-Barr virusgp350/220 DNA and amino acid sequences are found in articles by Beisel,J. Virology 54:665 (1985) and Biggin, EMBO J. 3:1083 (1984) and in U.S.Pat. No. 4,707,358 issued to Kieff, et al. (1987). A comparison of DNAsequences encoding gp350/220 in Epstein-Barr virus types A and B isdisclosed in Lees, Virology 195:578 (1993). Monoclonal antibodies thatexhibit neutralizing activity against gp350/220 glycoprotein of EBV aredisclosed in Thorley-Lawson, Proc. Natl. Acad. Sci. 77:5307 (1980).Lastly, splice site consensus sequences for donor and acceptor splicesites are disclosed in Mount, Nucleic Acids Res.10:459 (1982).

SUMMARY OF THE INVENTION

In one aspect this invention provides non-splicing variants of the EBVgp350/220 DNA sequence. The DNA sequences of the invention may includean isolated DNA sequence that encodes the expression of homogeneousgp350 protein. The DNA sequence coding for gp350 protein ischaracterized as comprising the same or substantially the samenucleotide sequence in FIG. 1 wherein the native nucleotides at thedonor and acceptor splice sites are replaced with non-nativenucleotides, and fragments thereof. This DNA sequence may include 5′ and3′ non-coding sequences flanking the coding sequence and further includean amino terminal signal sequence. FIG. 1 illustrates the non-codingsequences and indicates the end of the putative signal sequence with anasterisk. It is understood, however, that the DNA sequences of thisinvention may exclude some or all of these flanking or signal sequences.The non-splicing variant DNA sequences of the invention are produced byintroducing mutations into the FIG. 1 DNA sequence in the donor andacceptor splice sites of the gene encoding gp350/220. This eliminatesproduction of gp220 protein so that only the gp350 protein is produced.

Accordingly, in another aspect the invention comprises homogeneous gp350proteins, and methods of making the proteins by expression of thenon-splicing variant of EBV gp350/220 DNA sequence in an appropriateprokaryotic or eukaryotic host cell under the control of suitableexpression control sequence. As the term is used here with respect togp350 proteins, homogeneous means free or substantially free from gp220protein. We note that homogeneous gp350 protein, recombinantly producedin mammalian or insect cells, has not to our knowledge ever beenreported in the scientific literature heretofore.

In yet another aspect, homogeneous gp350 proteins, additionally havingdeletions resulting in a secreted product are provided. Such deletionscomprise either removal of the transmembrane region or removal of thetransmembrane region and the remaining C-terminus of gp350. Suchadditionally modified DNA sequences and the proteins encoded thereby areyet another aspect of this invention.

Also provided is a recombinant DNA molecule comprising vector DNA and aDNA sequence encoding homogeneous gp350 protein. The DNA moleculeprovides the gp350 sequence in operative association with a suitableregulatory sequence capable of directing the replication and expressionof homogeneous gp350 in a selected host cell. Host cells transformedwith such DNA molecules for use in expressing recombinant homogeneousgp350 are also provided by this invention.

The DNA molecules and transformed host cells of the invention areemployed in another aspect of the invention, a novel process forproducing recombinant homogeneous gp350 protein or fragments thereof. Inthis process a cell line transformed with a DNA sequence encoding ahomogeneous gp350 protein or fragment thereof (or a recombinant DNAmolecule as described above) in operative association with a suitableregulatory or expression control sequence capable of controllingexpression of the protein is cultured under appropriate conditionspermitting expression of the recombinant DNA. The expressed protein isthen harvested from the host cell or culture medium by suitableconventional means. The process may employ a number of known cells ashost cells; presently preferred are mammalian cells and insect cells.

The DNA sequences and proteins of the present invention are useful inthe production of therapeutic and immunogenic compounds having EBVantigenic determinants. Such compounds find use in subunit vaccines forthe prophylactic treatment and prevention of EBV related diseases, suchas mononucleosis, Burkitt's lymphoma and nasopharyngeal carcinoma.Accordingly, in yet another aspect the invention comprises suchtherapeutic and/or immunogenic pharmaceutical compositions forpreventing and treating EBV related conditions and diseases in humanssuch as infectitious mononucleosis, Burkett's lymphoma andnasopharyngeal carcinoma. Such therapeutic and/or immunogenicpharmaceutical compositions comprise a immunogenically inducingeffective amount of one or more of the homogeneous gp350 proteins of thepresent invention in admixture with a pharmaceutically acceptablecarrier such as aluminum hydroxide, saline and phosphate buffered salineas are known in the art. By “immunogenically inducing” we mean an amountsufficient for stimulating in a mammal the production of antibodies toEBV. Alternatively, the active ingredient may be administered in theform of a liposome-containing aggregate. For prophylactic use, suchpharmaceutical compositions may be formulated as subunit vaccines foradministration in human patients. Patients may be vaccinated with a dosesufficient to stimulate antibody formation in the patient; andrevaccinated after six months or one year.

A further aspect of the invention therefore is a method of treating EBVrelated diseases and conditions by administering to a patient,particularly to a human patient, an immunogenically inducingtherapeutically effective amount of a homogeneous gp350 protein in asuitable pharmaceutical carrier. Still another aspect of the inventionis a method of stimulating an immune response against EBV byadministering to a patient an immunogenically inducing effective amountof a homogeneous gp350 protein in a suitable pharmaceutical vehicle.

Other aspects and advantages of the invention are described further inthe following detailed description.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the DNA and amino acid sequence of gp350/220 (FromBeisel, J. Virology 54:665 (1985)). The donor and acceptor splice sitesare indicated. The transmembrane region is delineated with thehorizontal arrows and an asterisk (*) marks the end of the putativesignal sequence. Nucleotide numbering is shown at the left; amino acidnumbering at the right.

FIG. 2 illustrates construction of gp350 deletion and site directedmutants. The plasmid maps labelled pMDTM and pMSTOP exemplify thenon-splicing gp350/220 variants of the invention. In section (A), alinear model of the gp350 protein is shown approximately to scale withthe encoding clone, BLLF1, below. An N-terminal signal sequence (SS) andthe transmembrane domains (TM) are indicated on the protein andimportant restriction sites are indicated on the gene diagram. The gp350gene was cloned in two segments, the HindIII/BfaI BLSH1 fragment and theBanI/HindIII BLSH2 fragment. SCYT was created using the polymerase chainreaction from the region of BLLF1 indicated. In (B), the cloning schemefor pDTM, pSTOP, pMDTM, and pMSTOP is illustrated (plasmids not toscale). The details of the cloning are described in Examples 1 and 2.Plasmid maps are marked with the relevant restriction sites, the cloningvectors used and the gp350 gene fragments. Splice site mutations inpMDTM and pMSTOP are indicated by asterisks.

FIG. 3 illustrates the results of immunoprecipitation of homogeneousgp350 protein from pMDTM clones as analyzed by SDS-PAGE. Positivecontrol (GH3Δ19) cells secreting a truncated form of the gp350/220proteins, negative control (pEE14) cells and several pMDTM clones weremetabolically labeled with ³⁵S-methionine for 5.5 hours; homogeneousgp350 protein was immunoprecipitated from the resulting tissue culturesupernatants. For each cell type, samples of labeled tissue culturesupernatants (S) and gp350/220 precipitations (Ip) were electrophoresedon 5% SDS-PAGE (polyacrylamide gel electrophoresis). Location ofmolecular weight markers are indicated on the left side.

DETAILED DESCRIPTION

Disclosed are compositions and methods comprising cloned EBV DNAsequences encoding non-splicing variants of gp350 protein. As noted,such non-splicing variants are referred to herein as homogeneous gp350proteins. Normally, when the gp350/220 gene is expressed in mammaliancells two gene products are generated, gp350 and gp220, due to RNAsplicing of the gene. The invention allows for only one gene product,gp350, to be produced. The invention involves removing some or all ofthe RNA splice site signals in the gp350 gene and expressing the gene ina suitable host cell. Mutations in the gp350/220 gene were introduced toprevent production of the 220 kD version of the protein when thegp350/220 gene is expressed in mammalian cells. As a result, mRNAtranscripts encoding only gp350 are produced. The elimination of gp220expression by using a gp350/220 gene non-splicing variant will result inincreased production of gp350 relative to gp220. Production of gp220 isnot essential for production of an effective anti-EBV vaccine becausegp350 contains all the potential antigenic sites found on gp220.

Therefore, one aspect of this invention provides a DNA sequence encodinga polypeptide sequence substantially the same as gp350, except that thedonor splice site codon encoding amino acid 501 and the acceptor splicesite codon encoding amino acid 698 have been modified by replacement ofnative nucleotides with non-native nucleotides. Preferably the nativenucleotides are replaced with non-native nucleotides such that the aminoacid sequence remains the same. Specifically, in the example, nativenucleotides AAGT at the donor splice site (nucleotides 1500 through1504) and native nucleotides A and T flanking the GG acceptor splicesite (nucleotides 2091 and 2094) were replaced with nucleotides GTCA andT and A, respectively. Consequently, the Glutamine at amino acidposition 500 and the Serine at position 501 remained the same as aresult of this substitution in the donor site. Likewise, the Threonineat amino acid position 697 and the Glycine at position 698 remained thesame as a result of the modification in the acceptor site.

Analogously, substitutions other than those specifically exemplifiedcould readily be performed by one skilled in the art as is more fullydescribed below.

Therefore, in one aspect the invention comprises homogeneous gp350proteins. The homogeneous gp350 proteins are further characterized byhaving an amino acid sequence substantially the same as that shown inFIG. 1 from amino acids 1 through 907, from amino acids 1 through 862 orfrom amino acids 1 through 907 and excepting amino acids 863 through881, each with or without the N-terminal 18 amino acid signal sequence.In addition, analogs of homogeneous gp350 proteins are provided andinclude mutants in which there are variations in the amino acidssequence that retain antigenic activity and preferably have a homologyof at least 80%, more preferably 90%, and most preferably 95%, with thecorresponding region of the homogeneous gp350 proteins. Examples includeproteins and polypeptides with minor amino acid variations from theamino acid sequence of FIG. 1; in particular, conservative amino acidsreplacements. Conservative replacements are those that take place withina family of amino acids that are related in their side chains.Genetically encoded amino acids are generally divided into fourfamilies: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) non-polar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine. cysteine, serine, threonine, tyrosine.Phenylalanine, tryptophan and tyrosine are sometimes classified jointlyas aromatic amino acids. For example, it is reasonable to expect that anisolated replacement of a leucine or a similar conservative replacementof an amino acid with a structurally related amino acid will not have amajor effect on antigenic activity or functionality.

The invention offers the advantage of simpler purification of gp350.Because gp350 and gp220 have similar biochemical properties, gp220 isoften co-purified in preparations of gp350. Cells expressing only thenon-splicing variant of the gp350/220 gene simplifies proteinpurification. This will reduce the costs of producing gp350. Theinvention also makes biochemical characterization of the startingmaterial for gp350 purification easier. Because only one species ispresent, protein content analysis and amino acid sequence analysis maybe performed without accounting for the presence of a second species.

The invention additionally offers the advantage of increased gp350production. Prevention of gp350 gene splicing will shift the cell fromdual production of gp350 and gp220 to the production of gp350 alone. Insome cells, the concentrations of gp220 have been estimated to be30%-100% of the gp350 concentration. With the gene splicing eliminated,gp350 production will be increased by the lack of gp220 production.

The DNA sequence of the gp350/220 gene is described by Beisel, J.Virology 54:665 (1985) and Biggin, EMBO J. 3:1083 (1984) and isillustrated in FIG. 1. The gene is an open reading frame of 2721 bases,encoding 907 amino acids and specifying a primary translation product ofabout 95 kD. The difference between predicted and actual valuesrepresents extensive glycosylation of the protein. 591 bases (encoding197 amino acids) are spliced out to produce gp220. The apparentmolecular weight of gp350/220 gene products may also vary depending uponthe type of measurement system used, glycosylation site utilization indifferent cell types, post-translational processing differences orselective gene mutation. Measured values vary for the products ofdifferent gp350/220 gene non-splice site variants but the term“homogeneous gp350 protein or proteins” encompasses gene products of thenon-splicing variant, optionally having additional deletions ormutations such as the C-terminal deletions and/or transmembranemodifications also disclosed herein. The term “gp220 protein” refers tothe alternatively spliced gp350/220 gene product with a molecular weightof approximately 220 kD. Splice-sites in the gp350/220 gene wereidentified by comparison of the gp350/220 gene with consensus donor andacceptor splice sequences based on other genes, predominantly fromeukaryotic organisms. The consensus sequences developed by Mount,Nucleic Acids Res. 10:459 (1982) from studying the splice sites in othergenes are:

${Donor}:{\frac{A}{C}{{AG}/G^{*}}T^{*}\frac{A}{G}{AGT}}$${Acceptor}:{\frac{T}{C}N_{11}\frac{C}{T}A^{*}{G^{*}/G}}$

The bases asterisked above represent bases that appear in 100% of allsplice sites (highly conserved). Positions with two bases or one baserepresent conserved positions (non highlighted positions). The slashindicates the actual site of splicing.

In the gp350/220 gene the donor splice site occurs after nucleotide 1501and the acceptor splice occurs after nucleotide 2092, as shown by DNAsequencing (Biggin, EMBO J. 3:1083 (1984)) of the gp350/220 gene. (Thenumbering used herein and in FIG. 1 conforms to the numbering inBiggin). The splice site occurs in the corresponding gene region in theType B strain of EBV (the donor splice site after A₁₅₀₁ and the acceptorsplice site after G₂₀₂₉). The invention encompasses compositions madeusing either the A or B strain or another EBV strain's splice site toproduce a single species of mRNA from the gp350/220 gene. The DNAsequence of the Type A form of the virus from strain B95-8 was used inthe Examples although the DNA sequence of the Type B strain couldequally have been used, because the translated gene products of Type Aand B strains are 98% identical. The B strain lacks amino acids 507through 520 and 570 through 576. The type A strain was used because itcontains all the possible gp350 antigenic sites. Alternatively, EBVgp350/220 having strain-specific sequences could be used in accordancewith the teachings herein to produce EBV strain-specific homogeneousgp350 proteins having immunogenic properties specific to a particularstrain and therefore useful in immunogenic and/or therapeuticcompositions for the prevention or treatment of strain specific EBVrelated diseases. Table 1 shows the wild type nucleotide and amino acidsequences of the donor and acceptor splice sites.

To prevent RNA splicing of the gp350/220 gene, mutations were introducedinto the gp350/220 gene nucleic acid sequence to replace the relevantbase pairs of the RNA splice site. To render a splice-sitenonfunctional, preferably at least one of the bases out of the twohighly conserved bases framing the donor site or acceptor site should bereplaced with nonconserved bases, more preferably at least two highlyconserved bases should be mutated to nonconserved bases. Other conservedbases, more than two bases away from the splice site, can also bereplaced with nonconserved splice site bases to further decreaserecognition of the splice site. Both the donor and the acceptor site canbe changed to impair splicing mechanisms. Preferably, both the donor andthe acceptor contain at least one change each, in one of the four highlyconserved splice site base positions, and more preferably at least twochanges in two of the four highly conserved splice site base positions.If one splice site is not mutable due to a desire to maintain thewild-type amino acid sequence then it is preferable to introduce atleast two mutations to the other splice site.

Mutation at the gp350/220 splice sites may introduce changes into theamino acid sequence of the subsequently expressed gp350 protein.Preferably such changes should be conservative amino acid substitutions.Conservative substitutions in the amino acid sequence, as opposed tononconservative changes in the amino acid sequence, will help preserveantigenic sites. Conservative amino acid changes can be made as long asthe base change (or base changes) result in a suitable change in theinvariant donor/acceptor bases. For example, Gly could be substitutedfor Ser₅₀₁ at the donor splice site, using any Gly-specific codons otherthan GGU (use of GGU would preserve the G nucleotide and would notresult in the desired GT replacement in the splice signal). Likewise, atthe acceptor splice site, Gly₆₉₈ to Ala would be a conservative change,but since all Ala codons start with the highly conserved G nucleotide,this would not result in the desired replacement. Although Proline alsomight be a conservative amino acid change, proline would not be used toreplace a wild type amino acid because it would result in modificationof the tertiary structure of the protein and thereby mask one or moregp350 antigenic sites. Table 1 shows the acceptable conservative aminoacid replacements in the wild-type sequences. At the bottom of Table 1is an example of a mutation with conservative amino acid changes.

TABLE 1 Donor Acceptor Wild-type Sequences          splice      splice     GAA A|GT ACA G|GT      Glu Ser₅₀₁ Thr Gly₆₉₈       ↓   ↓  ↓   ↓Conservative a.a. changes      Asn Ala Ala Ser      Asp Gly Gly Thr     Gln Thr Ser ex.: GAC ACA TCG TCT      Asp Thr₅₀₁ Ser Ser₆₉₈

Although one aspect of the present invention comprises a non-splicingvariant of gp350/220, additional mutations of the gp350/220 codingsequence may also be desirable. In order to produce soluble homogeneousgp350 proteins (“soluble proteins” are either free in solution ormembrane associated but are not membrane integrated), for example, toavoid cell toxicity problems incurred by the expression of full lengthgp350 as an integral membrane protein, the membrane spanning region(also known as the transmembrane region) of gp350 is modified bydeletion of all or part of its encoding DNA sequence. The membranespanning region of gp350/220 comprises amino acids 861 (methionine)through 881 (alanine). See, Beisel, J. Virology 54:665 (1985).Preferably, at least 8 amino acids of the transmembrane region aredeleted, more preferably at least 12 amino acids are deleted and mostpreferably between 18 and 21 amino acids are deleted. Accordingly, inanother aspect, the invention provides non-splicing variants ofgp350/220 DNA and/or gp350 homogeneous protein additionally comprisingat least one deletion in the transmembrane region of the gp350/220 DNAand/or gp350 homogeneous protein that results in the expression ofsoluble homogeneous gp350 protein.

In addition to deleting all or part of the transmembrane domain of thenon-splicing gp350/220 variant, the C-terminal sequence following thetransmembrane domain and comprising amino acids 881 through 907 may alsobe deleted in whole or in part, as described herein, in accordance withthe invention. Thus, in another aspect the invention comprisesnon-splicing variants of gp350/220 DNA and/or homogeneous proteinfurther modified by deletion of all or a portion of the DNA encodingand/or amino acid sequence comprising the transmembrane region ofgp350/220 and even further modified by deletion of the remainingC-terminal DNA and/or amino acid sequences of gp350/220.

Accordingly, in another aspect the invention comprises non-splicingvariant DNA sequences encoding the homogeneous gp350 proteins of theinvention. Such DNA sequences comprise the DNA sequence of FIG. 1encoding amino acids 1 through 907 and further comprising the nucleotidesubstitutions taught herein to remove the donor and acceptor splicesites. Such DNA sequences optionally comprise truncated DNA sequences inwhich the nucleotides encoding all or part of the transmembrane domainand C-terminus comprising amino acids 861 through 907 are deleted anddeletion variants in which the nucleotides encoding all or part of thetransmembrane domain comprising amino acids 861 through 881 are deleted.The DNA sequences of the present invention encoding homogeneous gp350proteins may also comprise DNA capable of hybridizing under appropriatestringency conditions, or which would be capable of hybridizing undersuch conditions but for the degeneracy of the genetic code, to anisolated DNA sequence of FIG. 1. Accordingly, the DNA sequences of thisinvention may contain modifications in the non-coding sequences, signalsequences or coding sequences, based on allelic variation, speciesvariation or deliberate modification.

These non-splicing variant gp350/220 DNA sequences as disclosed hereincan be constructed using methods well known in the art. The modified DNAsequences of this invention can be expressed recombinantly, likewiseusing known methods, to produce the homogeneous gp350 proteins of thisinvention. Such recombinant proteins can be purified and incorporatedinto pharmaceutical compositions for the prophylactic treatment andprevention of EBV related diseases.

The non-splicing variants of gp350/220 DNA of this invention can beexpressed recombinantly in different types of cells using theappropriate expression control systems as is known in the art. Suitablecells known and available in the art include, but are not limited to,yeast cells such as Saccharomyces cerevisiae, bacterial cells such as E.coli and Bacillus subtilis and mammalian cells such as GH3, CHO, NSO,MDCK and C-127 cells. Vectors used with cell types are selected based ontheir compatibility with the cell type and expression control systemused. Cells and vectors that allow for the expression of secretedproducts of the gp350/220 gene are preferred. Typically for example, E.coli is transformed using derivatives of pBR322 which have been modifiedusing conventional techniques to contain the DNA sequences forexpression of the desired protein, in this instance the non-splicingvariant sequences of EBV gp350, with or without the sequences encodingthe C-terminus and/or membrane spanning region. pBR322 contains genesfor ampicillin and tetracycline resistance, which can be used asmarkers. See, Bolivar, Gene 2:95 (1977). Commonly used expressioncontrol sequences, i.e., promoters for transcription initiation andoptionally an operator or enhancer, include the beta-lactamase and lacpromoter systems (see Chang, Nature 198:1056 (1977)), the tryptophanpromoter system (see Goeddel, Nucleic Acids Res. 8:4057 (1980)) and thelambda-derived PL promoter and N-gene ribosome binding site (seeShimatake, Nature 292:128 (1981). However, any available promoter systemor expression control system that is compatible with prokaryotic hostcells can be used. Other exemplary host cells, plasmid and expressionvehicles are disclosed in U.S. Pat. Nos. 4,356,270 issued to Itakura(1982), 4,431,739 issued to Riggs (1984) and 4,440,859 issued to Rutter(1984).

Insect cells may also be used as host cells employing insect cellexpression. In the case of expression in insect cells, generally thecomponents of the expression system include a transfer vector, usually abacterial plasmid, which contains both a fragment of the baculovirusgenome, and a convenient restriction site for insertion of theheterologous gene or genes to be expressed; a wild type baculovirus witha sequence homologous to the baculovirus-specific fragment in thetransfer vector (this allows for the homologous recombination of theheterologous gene in to the baculovirus genome); and appropriate insecthost cells and growth media.

Currently, the most commonly used transfer vector for introducingforeign genes into AcNPV is pAc373. Many other vectors, known to thoseof skill in the art, have also been designed. These include, forexample, pVL985 (which alters the polyhedrin start codon from ATG toATT, and which introduces a BamHI cloning site 32 basepairs downstreamfrom the ATT; see Luckow and Summers, Virology (1989) 17:31.

The plasmid usually also contains the polyhedrin polyadenylation signal(Miller et al. (1988) Ann. Rev. Microbiol., 42:177) and a procaryoticampicillin-resistance (amp) gene and origin of replication for selectionand propagation in E. coli.

Baculovirus transfer vectors usually contain a baculovirus promoter. Abaculovirus promoter is any DNA sequence capable of binding abaculovirus RNA polymerase and initiating the downstream (5′ to 3′)transcription of a coding sequence (e.g. structural gene) into mRNA. Apromoter will have a transcription initiation region which is usuallyplaced proximal to the 5′ end of the coding sequence. This transcriptioninitiation region typically includes an RNA polymerase binding site anda transcription initiation site. A baculovirus transfer vector can alsohave a second domain called an enhancer, which, if present, is usuallydistal to the structural gene. Expression can be either regulated orconstitutive. For insect cell expression technology, see EP patentpublication 155 476.

Yeast, for example Saccharomyces cervisiae, may also be used as a hostcell. Various strains are available and may be used. Likewise, plasmidvectors suitable for yeast expression are known, as are promoter andexpression control systems. See for example, Myanohara, Proc. natl.Acad. Sci. 80:1 (1983)(PHO5 promoter), EP Patent Publication 012 873(leader sequences), Kurtz, Mol. Cell. Biol. 6:142 (1986), Ito, J.Bacteriol. 153:163 (1983) and Hinnen, Proc. Natl. Acad. Sci. 75:1929(1979)(transformation procedures and suitable vectors).

Eukaryotic cells from multicellular organisms may of course also be usedas hosts cells for the expression of genes encoding proteins andpolypeptides of interest. Useful host cell lines include VERO and HeLacells, and Chinese hamster ovary cells (CHO). Expression vectorscompatible with such cells are also available and typically includepromoters and expression control sequences, such as for example, theearly and late promoters from SV40 (see Fiers, Nature 273:113 (1978))and promoters from polyoma virus, adenovirus 2, bovine papilloma virusor avian sarcoma virus. Exemplary host cells, promoters, selectablemarkers and techniques are also disclosed in U.S. Pat. Nos. 5,122,469issued to Mather (1992), 4,399,216 issued to Axel (1983), 4,634,665issued to Axel (1987), 4,713,339 issued to Levinson (1987), 4,656,134issued to Ringold (1987), 4,822,736 issued to Kellems (1989) and4,874,702 issued to Fiers (1989).

Transformation of suitable host cells is accomplished using standardtechniques appropriate to such cells, such as CaCl₂ treatment forprokaryotes as disclosed in Cohen Proc. Natl. Acad. Sci. 69:2110 (1972)and CaPO₄ precipitation for mammalian cells as disclosed in Graham,Virology 52:546 (1978). Yeast transformation can be carried out asdescribed in Hsiao, Proc. Natl. Acad. Sci. 76:3829 (1979) or asdescribed in Klebe, Gene 25:333 (1983).

The construction of suitable vectors containing the non-splicing variantgp350 sequence (with or without the additional modifications disclosedhere resulting in deletion of the C-terminus and/or the membranespanning region) is accomplished using conventional ligation andrestriction techniques now well known in the art. Site specific DNAcleavage is performed by treating with suitable restriction enzyme(s)under standard conditions, the particulars of which are typicallyspecified by the restriction enzyme manufacturer. Polyacrylamide gel oragarose gel electrophoresis may be performed to size separate thecleaved fragments using standard techniques and the fragments bluntended by treatment with the Klenow fragment of E. coli polymerase I inthe presence of the four deoxynucleotide triphosphates. Treatment withS1 nuclease hydrolyzes any single-stranded portions. Syntheticoligonucleotides can be made using for example, thediethylphosphoamidite method known in the art. See U.S. Pat. No.4,415,732 (1983). Ligations can be performed using T4 DNA ligase understandard conditions and temperatures and correct ligations confirmed bytransforming E. coli or COS cells with the ligation mixture. Successfultransformants are selected by ampicillin, tetracycline or otherantibiotic resistance or using other markers as are known in the art.

Such recombinant DNA techniques are fully explained in the literature.See, e.g., Sambrook, MOLECULAR CLONING: A LABORATORY MANUAL, 2D ED.(1989); DNA CLONING, Vol. I and II (D N Glover ed 1985); OLIGONUCLEOTIDESYNTHESIS (M J Gait ed 1984); NUCLEIC ACID HYBRIDIZATION (B D Hames ed1984); TRANSCRIPTION AND TRANSLATION (B D Hames ed 1984); ANIMAL CELLCULTURE (R I Freshney ed 1986); B. Perbal, A PRACTICAL GUIDE TOMOLECULAR CLONING (1984); GENE TRANSFER VECTORS FOR MAMMALIAN CELLS (J RMiller ed 1987 Cold Spring Harbor Laboratory); Scopes, PROTEINPURIFICATION: PRINCIPLES AND PRACTICE, 2nd ed, (1987 Springer-Verlag NY) and HANDBOOK OF EXPERIMENTAL IMMUNOLOGY Vols 1-IV (EM Weired 1986).All such publications mentioned herein are incorporated by reference forthe substance of what they disclose.

Accordingly in another aspect the invention comprises vectors containingthe non-splicing variants of gp350/220 DNA sequences and host cells andfurther comprises a method of making a non-splicing variant of gp350/220protein by culturing said host cells containing a vector that iscarrying a non-splicing variant of a gp350/220 DNA sequence operativelylinked to an expression control sequence under culture conditionsenabling expression of the homogeneous gp350 protein.

The expressed homogeneous gp350 is purified from cell and culture mediumconstituents using conventional glycoprotein purification techniquessuch as, but not limited to, ultrafiltration, free flow electrophoresis,gel filtration chromatography, affinity chromatography, SDS-PAGE,differential NH₄SO₄ precipitation, lectin columns, ion exchange columnsand hydrophobicity columns as is known in the art. Small scaleanalytical preparations of gp350 are most readily purified usingSDS-PAGE or lectin affinity columns and such small scale preparationsfor use in vaccination or immune response experiments are most readilypurified using liquid chromatography. For large scale production ofcommercially significant quantities of gp350 for use in vaccinecompositions, a combination of ultrafiltration, gel filtration, ionexchange, and hydrophobic interaction chromatography are preferred.

The purified, homogeneous gp350 proteins of the present invention may beemployed in therapeutic and/or immunogenic compositions for preventingand treating EBV related conditions and diseases such as infectitiousmononucleosis, Burkitt's lymphoma and nasopharyngeal carcinoma. Suchpharmaceutical compositions comprise an immunogenically-inducingeffective amount of one or more of the homogeneous gp350 proteins of thepresent invention in admixture with a pharmaceutically acceptablecarrier, for example an adjuvant/antigen presentation system such asalum. Other adjuvant/antigen presentation systems, for instance, MF59(Chiron Corp.), QS-21 (Cambridge Biotech Corp.), 3-DMPL(3-Deacyl-Monophosphoryl Lipid A) (RibiImmunoChem Research, Inc.),clinical grade incomplete Freund's adjuvant (IFA), fusogenic liposomes,water soluble polymers or Iscoms (Immune stimulating complexes) may alsobe used. Other exemplary pharmaceutically acceptable carriers orsolutions are aluminum hydroxide, saline and phosphate buffered saline.The composition can be systemically administered, preferablysubcutaneously or intramuscularly, in the form of an acceptablesubcutaneous or intramuscular solution. Also inoculation can be effectedby surface scarification or by inoculation of a body cavity. Thepreparation of such solutions, having due regard to pH, isotonicity,stability and the like is within the skill in the art. The dosageregimen will be determined by the attending physician consideringvarious factors known to modify the action of drugs such as for example,physical condition, body weight, sex, diet, severity of the condition,time of administration and other clinical factors. Exemplary dosageranges comprise between about 1 μg to about 1000 μg of protein.

In practicing the method of treatment of this invention, animmunologically-inducing effective amount of homogeneous gp350 proteinis administered to a human patient in need of therapeutic orprophylactic treatment. An immunologically inducing effective amount ofa composition of this invention is contemplated to be in the range ofabout 1 microgram to about 1 milligram per dose administered. The numberof doses administered may vary, depending on the above mentionedfactors. The invention is further described in the following examples,which are intended to illustrate the invention without limiting itsscope.

Example 1 Deletion of the gp350/220 Transmembrane Region andTransmembrane Region Through C-Terminus to Create pDTM and pSTOP

The gp350/220 gene from the EBV B95-8 strain (Miller, et al., 1972), isavailable in a BamHI library as an open reading frame called BLLF1(Baer, Nature 310:207, 1984). To create the desired constructs (showndiagrammatically in FIG. 2B), the gp350/220 gene was cloned in twoparts: 1) BLSH1, a 2.3 kb HindIII/BfaI 3′ fragment and 2) BLSH2, a 337b.p. BanI/HindIII 5′ fragment (FIG. 2A). These fragments were clonedinto staging vectors so that the deletions of the C-terminal cytoplasmicand/or transmembrane-encoding domains could be performed. Because theBfaI site occurs at the 5′ end of the region encoding the gp350transmembrane (TM) domain, it was used to construct the TM domaindeletions and TM domain deletions with adjacent C-terminus deletions.Using BfaI, it was possible to create deletions retaining only two aminoacids of the TM region (Table 2).

1. Construction of pDTM From pSTG1, and pSTG3

The plasmid pDTM is comprised of a gp350/220 nucleic acid sequence thatlacks a complete TM coding region. This construct was made using twostaging vectors pSTG 1 and pSTG3. A 450 bp PCR product, SYCT, thatintroduced a BfaI site at the 3′ end of the TM region was made using aBLLF1 clone target sequence (FIG. 2). The PCR primers used are asfollows:

       BfaI Primer 1: GG ATC CTA GAC TGC GCC TTT AGG CGT A BLLF1:       ... GAC TGC GCC TTT AGG CGT A.. A.A.:       ... Asp Cys Ala Phe Arg Arg ...           ↑End of TM RegionPrimer 2: GGA TCC TCT GTT CCT TCT GCT CCA GTG BLLF1:... ... TCT GTT CCT TCT GCT CCA GTG

The BfaI site of Primer 1 was used to clone a BfaI/XmaI fragment of SCYTinto pSTG 1. The remainder of Primer 1 corresponds to the amino acidsequence encoded by clone BLLF1. Primer 2 corresponds to a regionoutside the gp350/220 open reading frame on the 3′ side of the gene. TheSCYT PCR fragment was cut with BfaI and XmaI to produce a 136 base pairfragment which was cloned into a pMT11 vector (Spaete and Mocarski,1985) along with a second fragment, a BLSH1 HindIII/BfaI fragment, tocreate pSTG1. Sequencing across the BfaI site indicated that all of theTM amino acid coding region was deleted except for amino acids Met andLeu (see Table 2). A third BLLF1 fragment, BLSH2, was cloned into pMT11to create pSTG3. A 16 base pair BanI/XbaI oligonucleotide linker outsideof the gp350/220 gene coding sequence was used to clone the BLSH2BanI/HindIII fragment into the pSTG3. A 2.4 HindIII/XmaI pSTG1 fragment,was cloned into a pEE14 vector (Celltech, England) together with a 0.3XbaI/HindIII pSTG3 fragment to complete the pDTM construct.

2. Construction of pSTOP Using Vectors pSTG2 and pSTG3

The plasmid pSTOP comprises a gp350/220 gene that lacks a TM region andthe C-terminal cytoplasmic region adjacent the TM region. To create thisconstruct, a 16 base pair BfaI/EcoRI oligonucleotide linker was createdwith stop codons (underlined) in three frames following the BfaI stickyend as shown below:

TAT AGA CTA GTC TAG G   A TCT GAT CAG ATC CTT AA

The 5′ overhang (TA) of the upper sequence is a sticky end for a BfaIrestriction site and the 5′ overhang (TTAA) of the lower sequence is anEcoRI sticky end. This 16 base pair linker was used to clone a BLSH1HindIII/BfaI fragment into pMT11, in order to create pSTG2. A 2.3 kbpSTG2 HindIII/EcoRI fragment and the pSTG3 0.3 kb XbaI/HindIII fragmentwere cloned into pEE14 to create pSTOP.

3. Comparison of the Wild-Type, pDTM and pSTOP Sequences at the TMRegion

The oligonucleotide sequence and translated amino acid sequence of thewild type, pSTOP, and pDTM 3′ ends of gp350 DNA and amino acid sequencesare shown in Table 2 below. Arrows indicate the beginning and end of thewild-type transmembrane domain (TM). Only two amino acids from thetransmembrane domain are retained in pDTM and pSTOP, Met₈₆₁ and Leu₈₆₂(see also FIG. 1). Note that a stop codon immediately follows Leu₈₆₂ inpSTOP. In pDTM the former location of the deleted transmembrane regionis marked “ΔTM”. (In the Table, the native amino acids are indicated.)

TABLE 2 3′ End of gp350 Wild-Type Sequence...AAC CTC TCC ATG CTA   GTA CTG. . . . .GTC ATG GCG GAC   TGC GCC...Asn Leu Ser Met Leu₈₆₂ Val Leu. . . . .Val Met Ala Asp₈₈₂ Cys Ala              ↑                                    ↑            TM start                             TM end 3′ End of pSTOP...AAC CTC TCC ATG CTA   TAG ACT AGT TCT AGG ......Asn Leu Ser Met Leu₈₆₂ STOP 3′ End of pDTM...AAC CTC TCC ATG CTA   GAC   TGC GCC......Asn Leu Ser Met Leu₈₆₂ Asp₈₈₂ Cys Ala...                       

                         ΔTM

Example 2 Removal of the gp350/220 Gene Donor and Acceptor Splice Sitesto Create pMDTM and pMSTOP

In order to obtain homogeneous production of a gp350 protein the highlyconserved and conserved bases of the gp350/220 gene splice site werechanged. Four bases were changed in the donor splice site, including thehighly conserved GT pair that occurs in 100% of all splice sites. Twoconserved donor site bases, AA, were replaced with GT. The two highlyconserved (invariant) donor splice site bases were changed from GT toCA. At the acceptor splice site, only one of the highly conservedacceptor splice site bases was altered to preserve the amino acidsequence. A second conserved acceptor splice site base was changed asindicated in Table 3. Table 3 summarizes the bases changed in the donorand acceptor splice sites of the gp350/220 gene.

TABLE 3 EBV gp350/220 Gene Splice Site Changes Donor Splice site: donordonor Wild-type: GAA A↓GT mutant: GAG* T*C*A* Glu Ser₅₀₁        Glu  Ser₅₀₁ Acceptor Splice site: acceptor acceptor Wild-type:ACA G↓GT mutant: ACT* GGA* Thr Gly₆₉₈         Thr  Gly₆₉₈

The bases changed by oligonucleotide-based mutagenesis are marked withan asterisk in the mutant sequences. The actual site of splicing isindicated by an arrow, and the encoded amino acids are shown. Note thatthe amino acid sequence does not change as a result of the nucleotidesubstitutions.

These nucleotide substitutions to the wild type gp350/220 donor splicesite and accepter splice site DNA sequences were accomplished usingoligonucleotide-mediated mutagenesis. A modified phage vector, M13TAC,was employed to produce mutations as described in Zoller, M. E. andSmith, M. (1983) Methods of Enzymol. 100:468. BamHI/XhoI fragments ofthe gp350/220 nucleotide sequence were cloned into the polylinker ofplasmid M13TAC using Asp718 and BanaHI restriction sites on thepolylinker, combined with a 19 bp oligonucleotide linker containingAsp718 and XhoI sticky ends. The plasmids M13DTM and M13STOP of Example1 (FIG. 2B), were used for the mutagenesis.

Two 42-mer oligonucleotides, PrDonor1 and PrAcceptor1, were made for usein the mutagenesis. Each was designed to be complementary to gp350/220gene sequences centering on either the donor or acceptor splice sites.The only region of the oligonucleotides that were not complementary tothe gp350/220 gene were the bases representing the desired mutations.Mutagenesis oligonucleotides comprised the following:

PrDonor1 Primer:GGT CAT GTC GGG GGC CTT TG |A CTC TGT GCC GTT GTC CCA TGG                        ** |* * EBV:GGT CAT GTC GGG GGC CTT AC |T TTC TGT GCC GTT GTC CCA TGG PrAcceptor1Primer: CTG TGT TAT ATT TTC ACC TC |C AGT TOG GTG AGC GGA 3GT TAG                        *  |  * EBV:CTG TGT TAT ATT TTC ACC AC |C TGT TGG GTG AGC GGA GGT TAG

The sequence of the mutagenesis oligonucleotides are labelled “Primer,”while the DNA sequence spanning the gp350/220 gene splice sites arelabelled “EBV.” Bases that were changed as a result of the mutagenesisare marked with an asterisk. The dashed line indicated the location ofthe splice.

The oligonucleotides PrDonor1 and PrAcceptor1 were hybridized tosingle-stranded clones of M13-DTM and M13-STOP. T4 DNA polymeraseholoenzyme was used to produce double-stranded M13 DNA and E. coli wastransformed with the double-stranded DNA. Using the vector M13TAC, anyclone that contained the desired mutation could be identified by a colorchange from white to blue in the presence of X-gal andisothiopropylgalactate. Blue plaques were picked and grown up, and DNAsequencing across splice junctions was used for the final identificationof mutant clones, labelled M13-MDTM and M13-MSTOP.

After identifying clones containing the desired mutations, BamHI/XhoIfragments were cut out of M13-MDTM and M13-MSTOP and ligated back intopDTM or pSTOP backbones to create the constructs pMDTM and pMSTOP,respectively. These constructs were transfected into CHO cells toexpress the non-splicing variant gp350/220 DNA sequences as described inExample 3.

Example 3 Expression of gp350 in CHO Cells

1. Transfection of gp350/220 Gene Constructs One method for producinghigh levels of homogeneous gp350 protein of the invention from mammaliancells involves the construction of cells containing multiple copies ofthe heterologous gp350 DNA sequence. The heterologous DNA sequence isoperatively linked to an amplifiable marker, in this example, theglutamine synthetase gene for which cells can be amplified usingmethionine sulphoximine. The pMDTM and pMSTOP vectors made in Example 2were transfected into CHO cells as discussed below, according to theprocedures of Crockett, Bio/Technology 8:662 (1990) and as described inthe Celltech Instruction Manual for the glutamine synthetase geneamplification system (1992).

CHO-K1 cells (ATCC CCR61) were maintained in glutamine-free EMEM (EaglesMinimal Essential Medium) supplemented with 10% fetal bovine serum, 100units/ml penicillin, 100 mg/ml streptomycin, MEM (Modified Eagle'sMedium) nonessential amino acids, and 1 mM sodium pyruvate (all obtainedfrom JRH Biosciences). The media was also supplemented with 60 mg/mlglutamic acid, 60 mg/ml asparagine, 7 mg/ml adenosine, 7 mg/mlguanosine, 7 mg/ml cytidine, 7 mg/ml uridine, and 2.4 mg/ml thymidine(all from Sigma.) This media preparation was used throughout thetransfection, with deviations from this recipe as noted.

One day prior to transfection 10-cm dishes were seeded with 3×10⁶ CHO-K1cells. On the day of transfection the cells were washed with 10 mlserum-free media per dish. Plasmid DNA (from the pMDTM, pMSTOP plasmids)was applied by CaPO₄ precipitation using conventional techniques. 10 ggsof each plasmid DNA precipitate was incubated with the CHO-K1 cells plus2 ml of serum-free media at 37° C. for 4.5 hours. Three replicates ofeach of the four plasmid DNA transfections were made. The cells werethen shocked for 1.5 minutes with 15% glycerol in HEPES-buffered saline.After rinsing with serum-free media, the cells were re-fed withserum-containing media and incubated for 24 hours.

The following day the media was changed to include 10% dialyzed fetalbovine serum (JRH Biosciences) and amplified by the addition of 25 μMmethionine sulphoximine (Sigma). Cells were re-fed with methioninesulphoximine-containing media every 3-5 days until the amplified cloneswere large enough for picking, approximately 13-14 days later. Cloneswere picked by scraping colonies off the dish with a sterile 200 μlpipetman tip and transferred to one well of a 96-well plate in mediawithout methionine sulphoximine. 1-2 days later the media was replacedwith media+25 μM methionine sulphoximine. After 4 days the culturesupernatants were harvested and assayed for protein products in an ELISAassay, as discussed below.

CHO cells were also transfected with the pEEl4 control vector alone(which contains no EBV sequences) and 24 clones of CHO-pEE14 were alsopicked and transferred to plates to serve as controls. (The controlclones were identified on the basis of survival in methioninesulphoximine.)

2. ELISA Assay

Following transfection, 241 clones of CHO-pMDTM and 158 clones ofCHO-pMSTOP were picked and grown up. Supernatants from these clones weretested for gp350 protein production. 96-well plates were coated withaffinity-purified rabbit anti-gp350/220 antibody (antibody MDP1; gift ofAndrew Morgan) diluted 1:2000 in 50 mM sodium borate buffer, pH 9. Theplates were incubated at 37° C. for 3-4 hours and washed 3 times withPBS+0.05% Tween 20 using a Nunc ImmunoWasher. After blotting dry, theplates were blocked by incubating with 2% BSA in PBS+0.01% Thimerosal at37° C. for 0.5 hours and washed again. Supernatants from the transfectedcells and control cells were added to the wells and incubated for 2hours at 37° C. The plates were then incubated with the primarydetection antibody, a mouse monoclonal antibody against gp350/220(antibody #C65221M; Biodesign International) at 1 mg/ml diluted in PBSwash buffer, 37° C. for 1 hour. After washing, the plates were incubatedwith the secondary antibody, horseradish peroxidase-conjugated goatF(ab)₂ fragments directed against mouse immunoglobulins (Human Igadsorbed; Biosource International.), 0.7 μg/ml in PBS+0.05% BSA and0.01% Thimerosal, at 37° C. for 1 hour. The plates were washed anddeveloped using ABTS (Pierce Chemicals) dissolved in Stable PeroxideSubstrate Buffer (Pierce Chemicals) for 0.5 hours at room temperature.The reaction was stopped with 1% SDS and the plates were read at 405 and650 nm wavelengths using a Molecular Devices Vmax ELISA plate reader. 24pMDTM and 18 pMSTOP clones tested positive for secreted gp350. Theclones exhibiting the highest ELISA signal were transferred to 24-wellplates for scale-up and further testing in a Western Blot and aradioimmunoprecipitation assay.

3. Western Blot and Radio Immunoprecipitation Assay

In an initial screening, tissue culture supernatants from the pMDTMtransfections were assayed for activity in a Western Blot. CHO cellsupernatants were purified on 5% SDS-PAGE gels, transferred tonitrocellulose overnight, and probed with anti-gp350 antibodies. SevenpMDTM clones were found to be positive for gp350 in the Western blotanalysis.

The pMDTM clones that were positive in the Western blot were furthertested by radioimmunoprecipitation for the presence of gp220. Selectedtransformed pMDTM cells, pEE14 control and GH3Δ19 control cells(described below) were grown overnight in six-well plates so that theywere approximately three-quarters confluent on the day of theexperiment. Each well contained approximately 5×10⁶ cells. Forlabelling, the media was removed from each well and replaced with 0.7 mlof methionine-free MEM (10% fetal calf serum)+100 μCi ³⁵S-methionine.The cells were incubated 5.5 hours at 37° C. and then microcentrifugedat 4000 rpm for 5 minutes. Homogeneous gp350 protein in the supernatantwas immunoprecipitated by addition of 10 μl of Sepharose-Protein A(Sigma) in a 50% slurry and 20 μl monoclonal anti-gp350/220 (antibody#C65221M, 100 mg/ml; Biodesign International), with overnight rocking at4° C. The mixture was then pelleted at 2000 rpm, 2 minutes at roomtemperature in a microcentrifuge and washed four times with severalvolumes of phosphate-buffered saline. After the final wash, all liquidwas removed from the pellet and replaced with 50 μl protein gel samplebuffer. The samples containing the precipitated immuno-complex wereboiled 5 minutes and run on a 5% SDS-PAGE. Immunoprecipitates werecompared to gel samples of tissue culture supernatants mixed 1:1 withprotein sample buffer. The gel was dried and autoradiographed withHyperfilm β-Max (Amersham).

FIG. 3 shows the autoradiographic results of SDS-PAGE analysis of theradioimmunoprecipitation. The cell line used as a positive control wasGH3Δ19 (gift of Elliot Keiff; Whang et al., 1987). GH3Δ19 cells secretea truncated form of the gp350/220 protein lacking the transmembrane andC-terminal cytoplasmic domains. For use as a negative control, CHO cellswere transfected with the pEEl4 vector alone and selected by methioninesulphoximine in parallel with the pMDTM transfection. In FIG. 3,supernatants (“S”) are shown in odd numbered lanes, alternated withimmunoprecipitates (“Ip”) shown in even numbered lanes. In control lane2, precipitation from the GH3Δ19 control cells results in two strongprotein bands at approximately 220 and 350 kD demonstrating productionof the truncated splice variant gp350 and gp220 proteins in about a 1:1ratio. As expected, these immunoprecipitated bands are concentrated withrespect to the radiolabelled tissue culture supernatant(non-immunoprecipitated sample) in lane 1. Also, as expected, no bandsare shown in the negative control (lane 4), since the pEEl4 vector doesnot contain any of the gp350/220 constructs.

SDS-PAGE analysis of the immunoprecipitation from supernatants of pMDTMclones in lanes 6, 8 and 10 results in a single strong band atapproximately 350 kD, the same as the higher molecular weight species inthe GH3Δ19 control lane 2. In contrast to the GH3Δ19 control lanehowever, an additional strong band at approximately 220 kD is absentfrom lanes 6, 8 and 10, although in lane 8 a very faint band migratingat a slightly lower molecular weight is revealed. This could represent adegradation product, a co-precipitated cellular product or a smallamount of gp220 protein resulting from a mistranslation or a mutationalevent that returns the deleted donor and acceptor splice sites to thenative nucleotide or amino acid sequences. Strong single bands atapproximately 350 kD were found in five other MTDM replicates tested(data not shown).

It is unlikely that the complete absence of the band at 220 kD in lanes6 and 10 is due to inefficient precipitation from MDTM supernatantssince in the ³⁵S-labelled GH3Δ19 control lane (2), a band at 220 kD iseasily visualized. Also, additional assays using the pDTM constructs ofExample 1 that contain the wild type splice sites result in two strongbands at 350 and 220 kD. Therefore, these results demonstrate thatdeletion of the splice sites results in production of gp350 protein inthe absence of production of gp220 protein.

This homogeneous gp350 protein, expressed in CHO cell lines, or in othermammalian or non-mammalian cell lines, can be further scaled up andhomogenous gp350 protein can be isolated and purified from conditionedmedium from the cell line using methods familiar in the art, includingtechniques such as lectin-affinity chromatography, reverse phase HPLC,FPLC, gel filtration and the like. See David, J. Immunol. Methods108:231 (1988) and Madej, Vaccine 10:777 (1992).

Example 4 Testing the Homogeneous gp350 Proteins for ImmunogenicActivity

The purified homogeneous gp350 proteins are incorporated intoappropriate vehicles for administration and administered to mice asfollows.

A 2×adjuvant-vehicle concentrate is prepared by mixing Pluronic L121 andsqualane in 0.4% (v/v) Tween 80 in phosphate buffered saline with (Thr)MDP in accordance with the procedure of David, J. Immunol. Methods108:231 (1988) and Allison, J. Immunol. Methods 95:157 (1986).

The composition for administration is prepared by addition of equalvolumes of protein and adjuvant-vehicle on the day of administration.The protein content should be with range of 5 micrograms to 50micrograms per dose.

BALB/c mice are immunized with three 0.1 ml intramuscular injections at0, 21 and 42 days. A pre-immunization bleed and successive bleeds taken10 days after each injection are obtained from the retro-orbital sinus.

Serum antibody levels are determined by an ELISA according to theprocedures described in Example 3. EBV neutralizing antibodies in thesera are quantified by their ability to inhibit transformation of fetalcord blood lymphocytes by EBV in vitro according to the methods of Moss,J. Gen. Virol. 17:233 (1972) and De Schryver, Int. J. Cancer 13:353(1974).

Alternatively, New Zealand white rabbits are inoculated by intramuscularadministration of five doses of protein emulsified in the foregoingadjuvant at 0, 21, 42, 63 and 84 days. The dose should be in the rangeof about 5 μg to 50 μg per inoculation. Sera is obtained two weeksfollowing the last dose and tested for antibody titers to the antigen,for cross-reactive antibody to viral gp350/220 from B95-8 cells and forin vitro EBV-neutralizing activity following the methods of Emini,Virology 166:387 (1988).

Because the ability of the EBV gp350/220 protein to induce protectiveimmunity in an animal model of EBV infection has already beenestablished, see Epstein, Clin. Exp. Immunol 63:485 (1986), similarpositive results from administration of a homogeneous gp350 proteincomposition are expected.

The disclosures of all publication identified herein are expresslyincorporated herein by reference. The foregoing detailed description isgiven for clearness of understanding only and no unnecessary limitationsare either understood or inferred therefrom, as modifications within thescope of the invention will be obvious to those skilled in the art.

1. An isolated DNA sequence that codes on expression for an homogeneousgp350 protein.
 2. A DNA sequence of claim 1 further characterized byhaving a nucleotide sequence in which at least one native nucleotideencoding serine at position 501 of FIG. 1 is replaced with a non-nativenucleotide and in which at least one native nucleotide encoding glycineat position 698 is replaced with a non-native nucleotide.
 3. A DNAsequence of claim 1 wherein said homogeneous gp350 protein is furthercharacterized by encoding an amino acid sequence having a deletion of atleast 8 amino acids in the transmembrane region.
 4. A DNA sequence ofclaim 3 wherein said homogeneous gp350 protein is further characterizedby encoding an amino acid sequence selected from the group consisting of(a) amino acid 19 through amino acid 862 of FIG.
 1. (b) amino acid 1through amino acid 862 of FIG.
 1. (c) amino acid 19 through amino acid862 and amino acid 882 through amino acid 907 of FIG.
 1. (d) amino acid1 through amino acid 862 and amino acid 882 through amino acid 907 ofFIG.
 1. 5. A vector comprising a DNA sequence of claim 1, claim 2, claim3 or claim
 4. 6. A host cell transformed with a DNA sequence of claim 1,claim 2, claim 3 or claim 4 in operative association with an expressioncontrol sequence capable of directing replicated and expression of saidDNA sequence.
 7. A process for producing a homogenous gp350 proteincomprising culturing a host cell of claim 6 in a suitable culture mediumand isolating said homogeneous gp350 protein from said cell. 8-11.(canceled)